*Target (protein/gene name): Protein Tyrosine Phosphatase B for Mycobacterium tuberculosis *NCBI Gene # or RefSeq#: 12501614 (Gene ID) *Protein ID (NP or XP #) or Wolbachia#: (pdb) 1U2Q *Organism (including strain): Mycobacterium tuberculosis *Etiologic Risk Group (see link below): NIAID category C priority pathogen *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis is a bacterial disease caused by the Mycobacterium tuberculosis bacterium which usually attacks the lungs but can affect other organs of the body as well. Tuberculosis is one of the world's deadliest and prolific diseases; in 2011 one third of the world became infected with Tuberculosis and 1.4 million of these cases resulted in death. Due to Tuberculosis' prolific nature, drug resistance has become a common problem, creating the need for new antibiotic drugs. http://www.cdc.gov/tb/ *Essentiality of this protein:Journal articles discussing essentiality of the tyrosine phosphatase in Mycobacterium tuberculosis: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1064030/
"Kinases and phosphatases are major families of enzymes involved in signal transduction and play key roles in intracellular event coordination."
"These two PTPases are released by M. tuberculosis into extracellular culture medium and are implicated in the interaction of mycobacteria with the host cell. Disruption of the MPtpB gene impairs the ability of M. tuberculosis to survive in guinea pigs and demonstrates the role of PTPases in the pathogenesis of disease caused by M. tuberculosis."
http://onlinelibrary.wiley.com/doi/10.1002/anie.200801566/full
"The causative organism of pulmonary tuberculosis, Mycobacterium tuberculosis, secretes two eukaryote-like protein tyrosine phosphatases, MptpA and MptpB, which selectively dephosphorylate human host proteins involved in interferon-γ signaling pathways, thereby preventing the initiation of host defense mechanisms.1 The inhibition of these enzymes might prevent the proliferation of Mycobacterium tuberculosis in human host macrophages and therefore represents a promising strategy for the development of selective antibiotics against this severe pathogen.2" *Complex of proteins?: yes, there are two forms (MptpA and MptpB) *Druggable Target: Druggability index (range: 0 to 1): 0.2 from TDRTargets website *EC#: 3.1.3.48 *Link to BRENDA EC# page:http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48 -- Show screenshot of BRENDA enzyme mechanism schematic
Natural enzyme mechanism for protein tyrosine phosphatase
Substitute substrate enzyme mechanism that will be used in the assay for protein tyrosine phoshatase
*Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric assay (at 405nm) using a substitute reagent, 4-ntrophenyl phosphate, and two cofactors, chloride ion and glycerol. --link to Sigma (or other company) page for assay or assay reagents (substrates): http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/5/t1196dat.Par.0001.File.tmp/t1196dat.pdf -- link (or citation) to paper that contains assay information: http://www.sciencedirect.com/science/article/pii/S0223523409000841
"with the absorbance at 405 nm measured to quantify the p-nitrophenol produced. The IC50 values of the inhibitors were determined by measuring the pNPP hydrolase activity of PTP1B in a range of different inhibitor concentrations."
1U2Q shown with glycerol and chloride ion in active site.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
1U2Q:A|PDBID|CHAIN|SEQUENCEMSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPS *length of your protein in Amino Acids: 168 residues *Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20.0266 kDa *Molar Extinction coefficient of your protein at 280 nm wavelength: 15720 (1/Mcm) *TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html): *CDS Gene Sequence (paste as text only):
TTGCGGCCGGGGCGGCTGTTCCGGTCCAGCGAGCTGAGCCGCCTCGACGACGCCGGCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGGAGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGTTCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCGCCGCACGAAACCGCATTCAAGAGGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGCCAGTCGATAAATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGAGCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACCCACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTCGGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGCAACGACTCCGTGCCACAACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGAGGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTACCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCGCGACGCCGGTATCAGCCAGGCCACAGTCAACCGGATGCGCGGGGTGCTGCTCGGATGA *GC% Content for gene: 65.21% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): TTGCGGCCGGGGCGGCTGTTCCGGTCCAGCGAGCTGAGCCGCCTCGACGACGCCGGCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGGAGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGTTCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCGCCGCACGAAACCGCATTCAAGAGGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGCCAGTCGATAAATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGAGCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACCCACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTCGGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGCAACGACTCCGTGCCACAACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGAGGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTACCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCGCGACGCCGGTATCAGCCAGGCCACAGTCAACCGGATGCGCGGGGTGCTGCTCGGATGA *GC% Content for gene (codon optimized): 67.71%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 12501614 (Gene ID)
*Protein ID (NP or XP #) or Wolbachia#: (pdb) 1U2Q
*Organism (including strain): Mycobacterium tuberculosis
*Etiologic Risk Group (see link below): NIAID category C priority pathogen
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis is a bacterial disease caused by the Mycobacterium tuberculosis bacterium which usually attacks the lungs but can affect other organs of the body as well. Tuberculosis is one of the world's deadliest and prolific diseases; in 2011 one third of the world became infected with Tuberculosis and 1.4 million of these cases resulted in death. Due to Tuberculosis' prolific nature, drug resistance has become a common problem, creating the need for new antibiotic drugs.
http://www.cdc.gov/tb/
*Essentiality of this protein: Journal articles discussing essentiality of the tyrosine phosphatase in Mycobacterium tuberculosis:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1064030/
"Kinases and phosphatases are major families of enzymes involved in signal transduction and play key roles in intracellular event coordination."
"These two PTPases are released by M. tuberculosis into extracellular culture medium and are implicated in the interaction of mycobacteria with the host cell. Disruption of the MPtpB gene impairs the ability of M. tuberculosis to survive in guinea pigs and demonstrates the role of PTPases in the pathogenesis of disease caused by M. tuberculosis."
http://onlinelibrary.wiley.com/doi/10.1002/anie.200801566/full
"The causative organism of pulmonary tuberculosis, Mycobacterium tuberculosis, secretes two eukaryote-like protein tyrosine phosphatases, MptpA and MptpB, which selectively dephosphorylate human host proteins involved in interferon-γ signaling pathways, thereby preventing the initiation of host defense mechanisms.1 The inhibition of these enzymes might prevent the proliferation of Mycobacterium tuberculosis in human host macrophages and therefore represents a promising strategy for the development of selective antibiotics against this severe pathogen.2"
*Complex of proteins?: yes, there are two forms (MptpA and MptpB)
*Druggable Target: Druggability index (range: 0 to 1): 0.2 from TDRTargets website
*EC#: 3.1.3.48
*Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48
-- Show screenshot of BRENDA enzyme mechanism schematic
*Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric assay (at 405nm) using a substitute reagent, 4-ntrophenyl phosphate, and two cofactors, chloride ion and glycerol.
--link to Sigma (or other company) page for assay or assay reagents (substrates): http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/5/t1196dat.Par.0001.File.tmp/t1196dat.pdf
-- link (or citation) to paper that contains assay information:
http://www.sciencedirect.com/science/article/pii/S0223523409000841
"with the absorbance at 405 nm measured to quantify the p-nitrophenol produced. The IC50 values of the inhibitors were determined by measuring the pNPP hydrolase activity of PTP1B in a range of different inhibitor concentrations."
http://www.ncbi.nlm.nih.gov/pubmed?cmd=retrieve&list_uids=7686722&dopt=citation
"These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at 282 nm or the fluorescence at 305 nm of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation."
-- List cost and quantity of substrate reagents and supplier:
glycerol from $36.60 (Sigma Aldrich) chloride ion and phosphatase (make in lab)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=glycerol&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286&mode=match%20partialmax
*Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1U2Q resolution-2.5A
-- For Homology Model option: N/A
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
*Current Inhibitors: There are seven inhibitors for protein tyrosine phosphatase A on BRENDA website.
http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.48&r=714454
http://www.sciencedirect.com/science/article/pii/S0968089610008898
This study found several potential ligands for protein tyrosine phosphatase B, but the IC50 data was relatively high.
http://onlinelibrary.wiley.com/doi/10.1002/anie.200801566/full
*Expression Information (has it been expressed in bacterial cells): The protein has been expressed using E.Coli cells. No trans-membrane regions.
http://www.ncbi.nlm.nih.gov/pubmed/15743966?dopt=Abstract
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser
*Purification Method: This protein can be modified with a HIS tag and purified in a column when bound to a nickle-bead complex.
*Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
1U2Q:A|PDBID|CHAIN|SEQUENCEMSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPS
*length of your protein in Amino Acids: 168 residues
*Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20.0266 kDa
*Molar Extinction coefficient of your protein at 280 nm wavelength: 15720 (1/Mcm)
*TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html):
*CDS Gene Sequence (paste as text only):
TTGCGGCCGGGGCGGCTGTTCCGGTCCAGCGAGCTGAGCCGCCTCGACGACGCCGGCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGGAGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGTTCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCGCCGCACGAAACCGCATTCAAGAGGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGCCAGTCGATAAATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGAGCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACCCACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTCGGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGCAACGACTCCGTGCCACAACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGAGGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTACCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCGCGACGCCGGTATCAGCCAGGCCACAGTCAACCGGATGCGCGGGGTGCTGCTCGGATGA
*GC% Content for gene: 65.21%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
TTGCGGCCGGGGCGGCTGTTCCGGTCCAGCGAGCTGAGCCGCCTCGACGACGCCGGCCGGGCAACGCTGCGCCGGCTGGGGATCACCGACGTTGCCGACCTGCGGTCGTCCCGGGAGGTTGCCCGCCGCGGTCCAGGACGGGTTCCGGACGGCATCGACGTCCACCTGCTGCCGTTCCCCGACCTCGCCGATGATGACGCCGACGACTCAGCGCCGCACGAAACCGCATTCAAGAGGCTGCTAACCAATGACGGGTCCAACGGCGAGTCCGGCGAATCCAGCCAGTCGATAAATGACGCGGCCACCCGCTACATGACCGACGAGTATCGCCAATTCCCAACGCGCAATGGAGCACAGCGCGCGCTACATCGTGTCGTCACACTGCTTGCCGCCGGACGCCCGGTGCTCACCCACTGCTTCGCGGGTAAGGATCGCACCGGCTTCGTGGTCGCGCTGGTGCTTGAAGCGGTCGGCCTGGACCGCGACGTCATCGTCGCCGACTACCTGCGCAGCAACGACTCCGTGCCACAACTGCGGGCCCGGATCTCCGAGATGATCCAGCAGCGTTTCGACACCGAACTGGCACCCGAGGTGGTGACGTTCACCAAGGCCCGGCTGTCCGACGGGGTCCTGGGTGTCCGCGCGGAGTACCTGGCCGCCGCACGCCAGACCATTGACGAGACCTACGGATCGCTGGGCGGCTACCTGCGCGACGCCGGTATCAGCCAGGCCACAGTCAACCGGATGCGCGGGGTGCTGCTCGGATGA
*GC% Content for gene (codon optimized): 67.71%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):